Combination of Anti-Clusterin Oligonucleotide with HSP90 Inhibitor for the Treatment of Prostate Cancer

ABSTRACT

The present invention provides a method for treating a mammalian subject affected by prostate cancer comprising i) an oligonucleotide which reduces clusterin expression and ii) a Heat Shock Protein 90 (Hsp90) inhibitor each in an amount that when in combination with the other is effective to treat the mammalian subject. The present invention also provides pharmaceutical compositions comprising an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor for use in treating a mammalian subject affected by prostate cancer. Also provided are oligonucleotides which reduce clusterin expression for use in combination with a Hsp90 inhibitor in treating a mammalian subject affected by prostate cancer, and a composition for treating a mammalian subject affected by prostate cancer comprising i) an oligonucleotide which reduces clusterin expression and ii) a Hsp90 inhibitor each in an amount that when in combination with the other is effective to treat the mammalian subject.

This application claims priority of U.S. Provisional Application No.61/453,102, filed Mar. 15, 2011, the contents of which are herebyincorporated by reference.

Throughout this application, various publications are referenced,including referenced in parenthesis. Full citations for publicationsreferenced in parenthesis may be found listed in alphabetical order atthe end of the specification immediately preceding the claims. Thedisclosures of all referenced publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The subject invention relates to combination therapy for treatingprostate cancer.

BACKGROUND OF THE INVENTION

Prostate cancer (PCa) is the most common cancer and the third mostcommon cause of cancer related mortality in men in the United States(Jemal et al., 2006). Androgen ablation remains the standard effectivetherapy for patients with advanced PCa, inhibiting proliferation andinducing apoptosis in tumor cells (Kyprianou et al., 1990).Unfortunately, after short-term remissions, surviving tumor cells recurwith castrate resistant prostate cancer (CRPC) and death usually within3 years in most men (Gleave et al., 1999). CRPC progression results frommechanisms attributed to re-activation of androgen receptor axis(Knudsen et al., 2009), alternative mitogenic growth factor pathways(Miyake et al., 2000; Culig et al., 2004), and stress-inducedprosurvival gene (Gleave et al., 1999; Miyake et al., 1999) andcytoprotective chaperone networks (Rocchi et al., 2004; Miyake et al.,2000). To significantly improve survival in men with PCa, newtherapeutic strategies to inhibit the appearance of this phenotype mustbe developed. It has been observed that numerous proteins are expressedin increased amounts by prostate tumor cells following androgenwithdrawal. At least some of these proteins are assumed to be associatedwith the observed apoptotic cell death which is observed upon androgenwithdrawal. (Raffo et al., 1995; Krajewska et al., 1996; McDonnell etal., 1992). The functions of many of the proteins, however, is notcompletely understood. Clusterin (also known as sulfated glycoprotein-2(SGP-2) or TRPM-2) is within this latter category.

Clusterin

Clusterin is a cytoprotective chaperone protein that promotes cellsurvival and confers broad-spectrum resistance to cancer treatments (Chiet al. 2005). In Sensibar et al., Cancer Research 55: 2431-2437, 1995,the authors reported on LNCaP cells transfected with a gene encodingclusterin, and watched to see if expression of this protein altered theeffects of tumor necrosis factor α (TNFα), to which LNCaP cells are verysensitive. Treatment of the transfected LNCaP cells with TNFα was shownto result in a transient increase in clusterin levels for a period of afew hours, but these levels had dissipated by the time DNA fragmentationpreceding cell death was observed.

As described in U.S. Pat. No. 7,534,773, the contents of which areincorporated by reference, enhancement of castration-induced tumor celldeath and delay of the progression of androgen-sensitive cancer cells toandrogen-independence may be achieved by inhibiting the expression ofclusterin by the cells.

Custirsen

Custirsen is a second-generation antisense oligonucleotide that inhibitsclusterin expression. Custirsen is designed specifically to bind to aportion of clusterin mRNA, resulting in the inhibition of the productionof clusterin protein. The structure of custirsen is available, forexample, in U.S. Pat. No. 6,900,187, the contents of which areincorporated herein by reference. A broad range of studies have shownthat custirsen potently regulates the expression of clusterin,facilitates apoptosis, and sensitizes cancerous human prostate, breast,ovarian, lung, renal, bladder, and melanoma cells to chemotherapy(Miyake et al. 2005), see also, U.S. Patent Application Publication No.2008/0119425 A1. In a clinical trial for androgen-dependent prostatecancer, the drugs flutamide and buserelin were used together incombination with custirsen, increasing prostate cancer cell apoptosis(Chi et al. 2004; Chi et al., 2005).

Hsp90

Heat shock protein 90 (Hsp90) is an ATPase-dependent molecular chaperonerequired for protein folding, maturation and conformationalstabilization of many “client” proteins (Young et al., 2000; Kamal etal., 2003). Hsp90 interacts with several proteins involved in CRPC,including growth factor receptors, cell cycle regulators and signalingkinases like Akt, androgen receptor (AR) or Raf-1, (Whitesell et al.,2005; Takayama et al., 2003). Tumor cells express higher Hsp90 levelscompared with benign cells (Kamal et al., 2003; Chiosis et al., 2003),and Hsp90 inhibition has emerged as an exciting target in CRPC and othercancers. Many Hsp90 inhibitors were developed targeting its ATP-bindingpocket, including natural compounds such as geldanamycin and itsanalogs, or synthetic compounds. These agents have been shown to inhibitHsp90 function and induce apoptosis in preclinical studies of colon,breast, PCa and other cancers (Kamal et al., 2003; Solit et al., 2003;Solit et al., 2002).

Combination Therapy

The administration of two drugs to treat a given condition, such asprostate cancer, raises a number of potential problems. In vivointeractions between two drugs are complex. The effects of any singledrug are related to its absorption, distribution, and elimination. Whentwo drugs are introduced into the body, each drug can affect theabsorption, distribution, and elimination of the other and hence, alterthe effects of the other. For instance, one drug may inhibit, activateor induce the production of enzymes involved in a metabolic route ofelimination of the other drug (Guidance for Industry. In vivo drugmetabolism/drug interaction studies—study design, data analysis, andrecommendations for dosing and labeling). Thus, when two drugs areadministered to treat the same condition, it is unpredictable whethereach will complement, have no effect on, or interfere with, thetherapeutic activity of the other in a human subject.

Not only may the interaction between two drugs affect the intendedtherapeutic activity of each drug, but the interaction may increase thelevels of toxic metabolites (Guidance for Industry. In vivo drugmetabolism/drug interaction studies—study design, data analysis, andrecommendations for dosing and labeling). The interaction may alsoheighten or lessen the side effects of each drug. Hence, uponadministration of two drugs to treat a disease, it is unpredictable whatchange will occur in the profile of each drug.

Additionally, it is difficult to accurately predict when the effects ofthe interaction between the two drugs will become manifest. For example,metabolic interactions between drugs may become apparent upon theinitial administration of the second drug, after the two have reached asteady-state concentration or upon discontinuation of one of the drugs(Guidance for Industry. In vivo drug metabolism/drug interactionstudies—study design, data analysis, and recommendations for dosing andlabeling).

Thus, the success of one drug or each drug alone in an in vitro model,an animal model, or in humans, may not correlate into efficacy when bothdrugs are administered to humans.

SUMMARY OF THE INVENTION

The present invention provides a method for treating a mammalian subjectaffected by prostate cancer comprising administering to the mammaliansubject i) an oligonucleotide which reduces clusterin expression and ii)a Heat Shock Protein 90 (Hsp90) inhibitor having the structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C₁-C₆)alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆)alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂ (C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or di-(C₁-C₁₀)                    alkylamino; and R₄ is optionally fused to a C₆-C₁₀                    aryl group, C₅-C₈ saturated cyclic group, or a                    C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅,    -   wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C₁-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₅-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₆) alkoxy, or                mono- or di-(C₁-C₁₀)alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆)alkyl; and    -   x is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, each in an amount that when in            combination with the other is effective to treat the            mammalian subject.

Hsp90 inhibitors having structure above are described in U.S. Pat. No.7,928,135, the entire contents of which are hereby incorporated hereinby reference.

The present invention provides a method for treating a mammalian subjectaffected by prostate cancer comprising administering to the mammaliansubject i) an oligonucleotide which reduces clusterin expression and ii)a Hsp90 inhibitor, which inhibitor is other than Hsp90i-1, each in anamount that when in combination with the other is effective to treat themammalian subject.

The present invention provides a method for treating a mammalian subjectaffected by prostate cancer comprising administering to the mammaliansubject i) an oligonucleotide which reduces clusterin expression and ii)a Hsp90 inhibitor which binds to Hsp90α and Hsp90β with a K_(a) of lessthan 50 nmol/L, or a prodrug thereof, each in an amount that when incombination with the other is effective to treat the mammalian subject.

The present invention provides a pharmaceutical composition comprisingan amount of an oligonucleotide which reduces clusterin expression, anda Hsp90 inhibitor having the structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C₁-C₆) alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆)alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₁-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂ (C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or                    di-(C₁-C₁₀)alkylamino; and R₄ is optionally fused to                    a C₆-C₁₀ aryl group, C₅-C₁₀ saturated cyclic group,                    or a C₄-C₁₀ heterocycloalkyl group; and        -   R₁ is optionally substituted at any available position with            C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀            alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, amino,            cyano, nitro, —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆) alkyl,            —SO₂NH₂, —SO₂NH(C₁-C₆) alkyl, —SO₂NH-aryl, —SO₂-aryl,            —SO—(C₁C₆)alkyl, —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy,            C₁-C₁₀ alkynyloxy, mono- or di-(C₁-C₁₀)alkylamino, —C₁-C₁₀            alkyl-Z, —OC₁-C₁₀ alkyl-Z, or R₅, wherein            -   Z is OR_(o) or —N(R₆)₂, wherein                -   each R₆ is independently —H or C₁-C₆ alkyl, or                    N(R₆)₂ represents pyrrolidinyl, piperidinyl,                    piperazinyl, azepanyl, 1,3- or 1,4-diazepanyl, or                    morpholinyl, each of which is optionally substituted                    with hydroxy, amino, aminoalkyl, C₁-C₆ alkyl, mono-                    or di(C₁-C₆) alkylamino, C₁-C₆ alkoxy, or halogen,                    and                -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                    alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and            -   R₅ is                -   (1) heteroaryl,                -   (2) aryl,                -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or                -   (4) saturated or unsaturated C₅-C₁₀                    heterocycloalkyl,                -    and                -   the R₅ groups are optionally substituted at least                    one group which is independently hydroxy, oxo, halo,                    amino, cyano, nitro, —SH, —S—(C₁-C₆) alkyl,                    —SO₂—(C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or                    di-(C₁-C₁₀)alkylamino;        -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or            halo(C₁-C₆) alkyl; and        -   X is N or CR₃, wherein            -   R₃ is H, halogen, or CH₃,                or a prodrug thereof, for use in treating a mammalian                subject affected by prostate cancer.

Hsp90 inhibitors having structure above are described in U.S. Pat. No.7,928,135, the entire contents of which are hereby incorporated hereinby reference.

The present invention provides a pharmaceutical composition comprisingan amount of an oligonucleotide which reduces clusterin expression, anda Hsp90 inhibitor, which inhibitor is other than Hsp90i-1, for use intreating a mammalian subject affected by prostate cancer.

The present invention provides a pharmaceutical composition comprisingan amount of an oligonucleotide which reduces clusterin expression, anda Hsp90 inhibitor which binds to Hsp90α and Hsp90β with a K_(a) of lessthan 50 nmol/L, or a prodrug thereof, for use in treating a mammaliansubject affected by prostate cancer.

The present invention provides an oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitorhaving the structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C₁-C₆) alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆)alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂ (C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or                    di-(C₁-C₁₀)alkylamino; and R₄ is optionally fused to                    a C₆-C₁₀ aryl group, C₅-C₈ saturated cyclic group,                    or a C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅,    -   wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C₁-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₅-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₆) alkoxy, or                mono- or di-(C₁-C₁₀) alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆)alkyl; and    -   X is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, in treating a mammalian subject            affected by prostate cancer.

Hsp90 inhibitors having structure above are described in U.S. Pat. No.7,928,135, the entire contents of which are hereby incorporated hereinby reference.

The present invention provides an oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitor,which inhibitor is other than Hsp90i-1, in treating a mammalian subjectaffected by prostate cancer.

The present invention provides an oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitor whichbinds to Hsp90α and Hsp90 with a K_(d) of less than 50 nmol/L, or aprodrug thereof, in treating a mammalian subject affected by prostatecancer.

The present invention provides a composition for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Hsp90 inhibitor having thestructure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C1-C₆)alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆)alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂(C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or                    di-(C₁-C₁₀)alkylamino; and R₄ is optionally fused to                    a C₆-C₁₀ aryl group, C₅-C₈ saturated cyclic group,                    or a C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅,    -   wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C₁-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₁-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₁₀) alkoxy, or                mono- or di-(C₁-C₁₀)alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆)alkyl; and    -   X is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, each in an amount that when in            combination with the other is effective to treat the            mammalian subject.

The present invention provides a composition for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Hsp90 inhibitor, whichinhibitor is other than Hsp90i-1, each in an amount that when incombination with the other is effective to treat the mammalian subject.

The present invention provides a composition for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Hsp90 inhibitor which bindsto Hsp90α and Hsp90β with a K_(a) of less than 50 nmol/L, or a prodrugthereof, each in an amount that when in combination with the other iseffective to treat the mammalian subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. Hsp90i-1 and Hsp90i-2 induce HSPs and clusterin (CLU)expression in prostate cancer (PCa) cells in vitro. PC-3 and LNCaP cellswere treated with 1 μM Hsp90i-2 (FIG. 1A) or 1 μM Hsp90i-1 (FIG. 1C) forthe indicated time points. In parallel, PC-3 and LNCaP cells weretreated for 48 h with Hsp90i-2 for the indicated doses (FIG. 1B).Protein extracts were analyzed for CLU, Hsp70, Akt and vinculin. Tumorcells were treated for 24 h with 1 μM Hsp90i-2 or 1 μM Hsp90i-1 (FIG.1D). mRNA extracts were analyzed by real-time PCR for CLU, Hsp90 andHsp70. ★★★, p<0.001.

FIGS. 2A-B. Hsp90i-2 induces HSP and CLU expression in PCa xenografts.Mice were treated for 6 weeks with 50 mg/kg Hsp90i-2-PRO (the prodrug ofHsp90i-2) or vehicle (Control). FIG. 2A, tumors were collected and CLUand Hsp70 were evaluated by immunohistochemical analysis. FIG. 2B, totalproteins were extracted from the xenograft tumors and CLU expression wasanalyzed by western blotting. The relative levels were normalized withGAPDH and estimated in densitometric units. ★★★, p<0.001.

FIGS. 3A-E. CLU induction following Hsp90 inhibitor treatment iscytoprotective via an increase of HSF-1 activity. FIG. 3A, LNCaP cellswere treated with indicated concentrations of Hsp90i-1 or Hsp90i-2 for48 h. FIG. 3B, LNCaP cells were transiently transfected with indicatedconcentrations of CLU-plasmid for 48 h. Total amount of plasmid DNAtransfected was normalized to 2 μg per well by the addition of an emptyvector. FIG. 3C (Top), LNCaP cells were transfected with 20 nM CLU siRNAor control siScr, followed of Hsp90i-1 or Hsp90i-2 treatment (1 μM) for48 h. FIG. 3D (bottom), LNCaP cells were treated twice with 300 nMcustirsen or control ScrB ASO. FIG. 3E, LNCaP and PC-3 cells weretreated twice with 300 nM custirsen or control ScrB ASO, followed by 1Mof Hsp90i-1 or Hsp90i-2 for 48 h. Cells were harvested, andHSE-luciferase activity or western blotting analyses were performed.Means of at least three independent experiments done in triplicate. ★★★,p<0.001; ★, p<0.05; ns, not significant.

FIGS. 4A-D. Increased potency of CLU knockdown and Hap90 inhibitorcombination treatment in PCa cells. FIG. 4A, LNCaP cells were treatedtwice with 300 nM custirsen or control ScrB ASO, followed by theindicated concentration of Hsp90i-1 or Hsp90i-2 for 48 h. Cell growthwas determined by crystal violet compared with control. FIG. 4B, dosedependent effects and combination index (CI) values calculated byCalcuSyn software were assessed in LNCap cells treated for 48 h withcustirsen alone, Hsp90i-2 alone or combined treatment at indicatedconcentration with constant ratio design between both drugs. The CI forED₅₀ and ED₇₅ was 0.4 and 0.75, respectively, indicative of acombination effect of this combined treatment. FIG. 4C and FIG. 4D,LNCaP cells were treated twice with 300 nM custirsen or control ScrB,followed by 1 μm Hsp90i-1 or Hsp90i-2 for 48 h. Cells were harvested,and western blotting analyses were performed (C). The proportion ofcells in subG1, G0-G1, S, G2-M was determined by propidium iodidestaining and caspase-3 activity was determined on the cell lysates andthe results are expressed in arbitrary units and corrected for proteincontent (D). All experiments were repeated at least thrice. $$$,p<0.001; ***, p<0.001; **, p<0.01*, p<0.05.

FIGS. 5A-C. Increased potency of custirsen+Hsp90i-1 combination in PC-3xenograft model. Mice were treated IP with 25 mg/kg Hsp90i-1 and 15mg/kg custirsen starting when tumors reached 300 mm as described inExample 6. FIG. 5A, the mean tumor volume of mice custirsen+Hsp90i-1 wascompared with control ScrB ASO+Hsp90i-1±SEM (n=7). ★★, p<0.01. FIG. 5B,in Kaplan-Meier curve, cancer-specific survival was compared betweenmice treated with custirsen+Hsp90i-1 and control ScrB ASO+Hsp90i-1 overa 72-d period. ★, p<0.05. FIG. 5C, tumors were collected after 72-d andCLU, Ki67 and TUNEL were evaluated by immunohistochemical analysis(original magnification: ×200).

FIGS. 6A-D. Increased potency of custirsen+Hsp90i-2-PRO combination inLNCaP xenograft model. Mice were treated with 25 mg/kg Hsp90i-2-PRO and15 mg/kg custirsen starting when serum PSA values relapsed topre-castration levels. The mean tumor volume (FIG. 6A) and the serum PSAlevel (FIG. 6B) were compared between the 4 groups±SEM (n=10). ★★★,p<0.001. FIG. 6C, PSA doubling time and velocity were calculated asdescribed in Example 6. ★, p<0.05. FIG. 6D, in Kaplan-Meier curve,cancer-specific survival was compared between the 4 groups over a 62-dperiod. ***, p<0.001. Progression-free survival was defined as time forthe first tumor volume doubling.

FIGS. 7A-B. Increased potency of custirsen+Hsp90i-2-PRO combinationtreatment apoptosis levels in CRPC LNCaP tumors. FIG. 7A, tumors werecollected after 57 days and CLU, Ki67, AR, AKT and TUNEL were evaluatedby immunohistochemical analysis (original magnification: ×200). FIG. 7B,total proteins were extracted from the xenograft tumors and CLU, AR, Aktand PSA were analyzed by western blotting. The relative levels werenormalized with vinculin and estimated in densitometric units±SEM.

FIGS. 8A-C. Clusterin protects tumor cells to Hsp90 inhibitors via aregulation of HSF-1. FIG. 8A, PC-3 cells were transfected to overexpressCLU compared to wt-PC-3 and treated with indicated concentrations ofHsp90i-2 for 48 h. Cell growth was determined by crystal violet andcompared with control. ★★, p≦0.01. FIG. 8B, tumor cells were treatedwith 20 nM HSF-1 siRNA vs control Scr siRNA and treated with μM Hsp90i-2for 48 h. Total proteins were extracted and, western blotting andcaspase 3/7 activity were performed. FIG. 8C, PC-3 cells were treatedwith 20 nM CLU siRNA vs control Scr siRNA and treated with 1 μM Hsp90i-1for 24 h. HSF-1 localization was assessed by immunofluorescencestaining.

FIG. 9. LNCaP and PC-3 cells were treated twice with 300 nM custirsen orcontrol ScrB ASO, followed by 1 μM of Hsp90i-1 or Hsp90i-2 for 48 h.Cells were harvested, and HSE-luciferase activity or western blottinganalyses were performed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating a mammalian subjectaffected by prostate cancer comprising administering to the mammaliansubject i) an oligonucleotide which reduces clusterin expression and ii)a Heat Shock Protein 90 (Hsp90) inhibitor having the structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C1-C₆) alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆) alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂ (C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or                    di-(C₁-C₁₀)alkylamino; and R₄ is optionally fused to                    a C₆-C₁₀ aryl group, C₅-C₈ saturated cyclic group,                    or a C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅,    -   wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C₁-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₅-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₆) alkoxy, or                mono- or di-(C₁-C₁₀)alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆)alkyl; and    -   x is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, each in an amount that when in            combination with the other is effective to treat the            mammalian subject.

The present invention provides a method for treating a mammalian subjectaffected by prostate cancer comprising administering to the mammaliansubject i) an oligonucleotide which reduces clusterin expression and ii)a Hsp90 inhibitor, which inhibitor is other than Hsp90i-1, each in anamount that when in combination with the other is effective to treat themammalian subject.

The present invention provides a method for treating a mammalian subjectaffected by prostate cancer comprising administering to the mammaliansubject i) an oligonucleotide which reduces clusterin expression and ii)a Hsp90 inhibitor which binds to Hsp90α and Hsp90β with a K_(a) of lessthan 50 nmol/L, or a prodrug thereof, each in an amount that when incombination with the other is effective to treat the mammalian subject.

In some embodiments, the Hsp90 inhibitor binds to Hsp900 and/or Hsp90with a K_(d) of less than about 70, 60, 50, 40, 35, 30, 25, 20, 15, 10,or 5 nmol/L.

In some embodiments, the cancer is androgen-independent prostate cancer.

In some embodiments, the amount of the oligonucleotide and the amount ofthe Hsp90 inhibitor when taken together is more effective to treat thesubject than when each agent is administered alone.

In some embodiments, the amount of the oligonucleotide in combinationwith the amount of the Hsp90 inhibitor is less than is clinicallyeffective when administered alone.

In some embodiments, the amount of the Hsp90 inhibitor in combinationwith the amount of the oligonucleotide is less than is clinicallyeffective when administered alone.

In some embodiments, the amount of the oligonucleotide and the amount ofthe Hsp90 inhibitor when taken together is effective to reduce aclinical symptom of prostate cancer in the subject.

In some embodiments, the mammalian subject is human.

In some embodiments, the oligonucleotide is an antisenseoligonucleotide.

In some embodiments, the antisense oligonucleotide spans either thetranslation initiation site or the termination site ofclusterin-encoding mRNA.

In some embodiments, the antisense oligonucleotide comprises nucleotidesin the sequence set forth in SEQ ID NO: to 11.

In some embodiments, the antisense oligonucleotide comprises nucleotidesin the sequence set forth in SEQ ID NO: 3.

In some embodiments, the antisense oligonucleotide is modified toenhance in vivo stability relative to an unmodified oligonucleotide ofthe same sequence.

In some embodiments, the oligonucleotide is custirsen.

In some embodiments, the amount of custirsen is less than 640 mg.

In some embodiments, the amount of custirsen is less than 480 mg.

In some embodiments, the amount of custirsen is administeredintravenously once in a seven day period.

In some embodiments, the amount of the Hsp90 inhibitor is less than 50mg/kg.

In some embodiments, the amount of the Hsp90 inhibitor is 25 mg/kg orless.

In some embodiments, the Hsp90 inhibitor is Hsp90i-2.

In some embodiments, a prodrug of the Hsp90 inhibitor is administered tothe mammalian subject which prodrug is Hsp90i-2-PRO.

In some embodiments, a prodrug of the Hsp90 inhibitor is administered tothe mammalian subject which prodrug is Hsp90i-2-PRO2.

In some embodiments, the combination of the oligonucleotide and theHsp90 inhibitor is effective to inhibit the proliferation of prostatecancer cells.

The present invention provides a pharmaceutical composition comprisingan amount of an oligonucleotide which reduces clusterin expression, anda Hsp90 inhibitor having the structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C₁-C₆)alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆)alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂(C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or di-(C₁-C₁₀)                    alkylamino; and R₄ is optionally fused to a C₆-C₁₀                    aryl group, C₅-C₈ saturated cyclic group, or a                    C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅,    -   wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C₁-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₁-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₅-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₆) alkoxy, or                mono- or di-(C₁-C₁₀)alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆)alkyl; and    -   X is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, for use in treating a mammalian            subject affected by prostate cancer.

The present invention provides a pharmaceutical composition comprisingan amount of an oligonucleotide which reduces clusterin expression, anda Hsp90 inhibitor, which inhibitor is other than Hsp90i-1, for use intreating a mammalian subject affected by prostate cancer.

The present invention provides a pharmaceutical composition comprisingan amount of an oligonucleotide which reduces clusterin expression, anda Hsp90 inhibitor which binds to Hsp90α and Hsp90β with a K_(a) of lessthan 50 nmol/L, or a prodrug thereof, for use in treating a mammaliansubject affected by prostate cancer.

The present invention provides an oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitorhaving the structure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃—C, cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C₁-C₆)alkylamino, nitro, halo(C₁-C₆)alkyl,        halo(C₁-C₆)alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            0 atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO_(z) (C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or di-(C₁-C₆)                    alkylamino; and R₄ is optionally fused to a C₆-C₁₀                    aryl group, C₅-C₈ saturated cyclic group, or a                    C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅,    -   wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C₁-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₅-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₆) alkoxy, or                mono- or di-(C₁-C₁₀) alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆) alkyl; and    -   X is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, in treating a mammalian subject            affected by prostate cancer.

The present invention provides an oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitor,which inhibitor is other than Hsp90i-1, in treating a mammalian subjectaffected by prostate cancer.

The present invention provides an oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitor whichbinds to Hsp90α and Hsp90β with a K_(a) of less than 50 nmol/L, or aprodrug thereof, in treating a mammalian subject affected by prostatecancer.

The present invention provides a composition for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Hsp90 inhibitor having thestructure:

or a pharmaceutically acceptable salt thereof, wherein

-   -   R₁ is H, C₁-C₁₄ alkyl, C₁-C₁₀ haloalkyl, C₃-C₇ cycloalkyl,        heterocycloalkyl, C₁-C₆ acyl, aryl, or heteroaryl, wherein each        alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group        is optionally substituted with from 1-4 groups that are        independently C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy,        amino, mono- or di-(C₁-C₆) alkylamino, nitro, halo(C₁-C₆) alkyl,        halo(C₁-C₆) alkoxy, or carboxamide, wherein        -   when R₁ is a C₁-C₁₄ alkyl group, up to five of the carbon            atoms in the alkyl group are optionally replaced            independently by R₄, carbonyl, ethenyl, ethynyl or a moiety            selected from N, O, S, SO₂, or SO, with the proviso that two            O atoms, two S atoms, or an O and S atom are not immediately            adjacent each other, wherein            -   R₄ is                -   (i) heteroaryl,                -   (ii) aryl,                -   (iii) saturated or unsaturated C₃-C₁₀ cycloalkyl, or                -   (iv) saturated or unsaturated C₂-C₁₀                    heterocycloalkyl,                -    wherein                -   each aryl, heteroaryl, saturated or unsaturated                    cycloalkyl, or saturated or unsaturated                    heterocycloalkyl, independently, is optionally                    substituted with at least one group, which                    independently is hydroxy, halo, amino, cyano,                    carboxy, carboxamido, nitro, oxo, —S—(C₁-C₆) alkyl,                    —SO₂ (C₁-C₆) alkyl, —SO₂-aryl, —SO—(C₁-C₆) alkyl,                    —SO-aryl, —SO₂NH₂, —SO₂NH—(C₁-C₆) alkyl,                    —SO₂NH-aryl, (C₁-C₆) alkoxy, or mono- or di-(C₁-C₁₀)                    alkylamino; and R₄ is optionally fused to a C₆-C₁₀                    aryl group, C₅-C₈ saturated cyclic group, or a                    C₄-C₁₀ heterocycloalkyl group; and    -   R₁ is optionally substituted at any available position with        C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro,        —SH, —S—(C₁-C₆)alkyl, —SO₂—(C₁-C₆)alkyl, —SO₂NH₂,        —SO₂NH(C₁-C₆)alkyl, —SO₂NH-aryl, —SO-aryl, —SO—(C1-C6)alkyl,        —SO₂-aryl, C₁-C₆ alkoxy, C₂-C₁₀ alkenyloxy, C₂-C₁₀ alkynyloxy,        mono- or di-(C₁-C₁₀)alkylamino, C₁-C₁₀ alkyl-Z, —OC₁-C₁₀        alkyl-Z, or R₅, wherein        -   Z is OR_(o) or —N(R₆)₂, wherein            -   each R₆ is independently —H or C₁-C₆ alkyl, or N(R₆)₂                represents pyrrolidinyl, piperidinyl, piperazinyl,                azepanyl, 1,3- or 1,4-diazepanyl, or morpholinyl, each                of which is optionally substituted with hydroxy, amino,                aminoalkyl, C1-C₆ alkyl, mono- or di(C₁-C₆) alkylamino,                C₁-C₆ alkoxy, or halogen, and            -   R_(o) is —H, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀                alkynyl, aryl, heteroaryl, or —C₁-C₆ acyl; and        -   R₅ is            -   (1) heteroaryl,            -   (2) aryl,            -   (3) saturated or unsaturated C₅-C₁₀ cycloalkyl, or            -   (4) saturated or unsaturated C₅-C₁₀ heterocycloalkyl,                -   and            -   the R₅ groups are optionally substituted at least one                group which is independently hydroxy, oxo, halo, amino,                cyano, nitro, —SH, —S—(C₁-C₆) alkyl, —SO₂—(C₁-C₆) alkyl,                —SO₂-aryl, —SO—(C₁-C₆) alkyl, —SO-aryl, —SO₂NH₂,                —SO₂NH—(C₁-C₆) alkyl, —SO₂NH-aryl, (C₁-C₆) alkoxy, or                mono- or di-(C₁-C₁₀) alkylamino;    -   R₂ is H, Cl, halogen, CF₃, CHF₂, CH₃, C₁-C₁₀ alkyl, or        halo(C₁-C₆) alkyl; and    -   X is N or CR₃, wherein        -   R₃ is H, halogen, or CH₃,            or a prodrug thereof, each in an amount that when in            combination with the other is effective to treat the            mammalian subject.

The present invention provides a composition for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Hsp90 inhibitor, whichinhibitor is other than Hsp90i-1, each in an amount that when incombination with the other is effective to treat the mammalian subject.

The present invention provides a composition for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Hsp90 inhibitor which bindsto Hsp90α and Hsp90β with a K_(a) of less than 50 nmol/L, or a prodrugthereof, each in an amount that when in combination with the other iseffective to treat the mammalian subject.

In some embodiments, the combination of the oligonucleotide and theHsp90 inhibitor is effective to inhibit the proliferation of prostatecancer cells.

In some embodiments, Hsp90 inhibitor-mediated induction of clusterinexpression is attenuated by custirsen, wherein the combination of theHsp90 inhibitor and custirsen delays the progression of CRPC. In someembodiments, the combination of the Hsp90 inhibitor and custirseninhibits tumor growth in the mammalian subject. In some embodiments, thecombination of the Hsp90 inhibitor and custirsen prolongs the survivalof the mammalian subject.

An aspect of the invention provides pharmaceutical compositioncomprising an amount of an oligonucleotide which reduces clusterinexpression, and a Hsp90 inhibitor for use in treating a mammaliansubject affected by prostate cancer.

An aspect of the invention provides oligonucleotide which reducesclusterin expression for use in combination with a Hsp90 inhibitor intreating a mammalian subject affected by prostate cancer.

An aspect of the invention provides a composition for treating amammalian subject affected by prostate cancer comprising i) anoligonucleotide which reduces clusterin expression and ii) a Hsp90inhibitor each in an amount that when in combination with the other iseffective to treat the mammalian subject.

Aspects of the invention involve the increased potency of a combinationtreatment comprising an oligonucleotide that targets clusterinexpression and an Hsp90 inhibitor compared to oligonucleotide or Hsp90inhibitor monotherapy. In some embodiments of the invention, thecombination of an oligonucleotide that targets clusterin expression andan HSP90 inhibitor increases prostate cancer cell apoptosis and/ordecreases prostate cancer cell proliferation compared to oligonucleotideor Hsp90 inhibitor monotherapy. In some embodiments, the combination ofan oligonucleotide that targets clusterin expression and an Hsp90inhibitor decreases the protein expression and/or a function of HSF-1compared to oligonucleotide or Hsp90 inhibitor monotherapy.

Aspects of the invention provide targeted strategies employing anoligonucleotide which reduces clusterin expression in combination withHsp90 inhibitors to improve patient outcome in castration-resistantprostate cancer.

The present invention relates to a method for treating a mammaliansubject affected by prostate cancer comprising i) an oligonucleotidewhich reduces clusterin expression and ii) a Heat Shock Protein 90(Hsp90) inhibitor, each in an amount that when in combination with theother is effective to treat the mammalian subject.

In some embodiments, the Hsp90 inhibitor is Hsp90i-1.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg/kg/day” includes 0.2 mg/kg/day, 0.3mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day etc. up to 5.0mg/kg/day.

Terms

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

As used herein, “about” in the context of a numerical value or rangemeans±10% of the numerical value or range recited or claimed.

As used in the specification and claims of this application, the term“clusterin” refers to a glycoprotein present in mammals, includinghumans, and denominated as such in the humans. The sequences of numerousclusterin species are known. For example, the sequence of humanclusterin is described by Wong et al., Eur. J. Biochem. 221 (3), 917-925(1994), and in NCBI sequence accession number NM_001831 (SEQ ID NO: 43).In this human sequence, the coding sequence spans bases 48 to 1397.

As used herein, “oligonucleotide which reduces clusterin expression” isan oligonucleotide with a sequence which is effective to reduceclusterin expression in a cell. The oligonucleotide which reducesclusterin expression may be, for example, an antisense oligonucleotideor an RNA interference inducing molecule.

As used herein, “antisense oligonucleotide” refers to a non-RNAioligonucleotide that reduces clusterin expression and that has asequence complementary to clusterin mRNA. Antisense oligonucleotides maybe antisense oligodeoxynucleotides (ODN). Exemplary sequences which canbe employed as antisense molecules in the invention are disclosed in PCTPatent Publication WO 00/49937, U.S. Patent PublicationUS-2002-0128220-A1, and U.S. Pat. No. 6,383,808, all of which areincorporated herein by reference. Specific antisense sequences are setforth in the present application as SEQ ID NOs: 1 to 11, and may befound in Table 1.

TABLE 1  Sequence Identification Numbers for Antisense OligonucleotidesSBQ ID NO: Sequence 1 GCACAGCAGG AGAATCTTCA T 2 TGGAGTCTTT GCACGCCTCG G3 CAGCAGCAGA GTCTTCATCA T 4 ATTGTCTGAG ACCGTCTGGT C 5CCTTCAGCTT TGTCTCTGAT T 6 AGCAGGGAGT CGATGCGGTC A 7ATCAAGCTGC GGACGATGCG G 8 GCAGGCAGCC CGTGGAGTTG T 9TTCAGCTGCT CCAGCAAGGA G 10 AATTTAGGGT TCTTCCTGGA G 11GCTGGGCGGA GTTGGGGGCC T

The ODNs employed may be modified to increase the stability of the ODNin vivo. For example, the ODNs may be employed as phosphorothioatederivatives (replacement of a non-bridging phosphoryl oxygen atom with asulfur atom) which have increased resistance to nuclease digestion. MOE(2′-O-(2-methoxyethyl)) modification (ISIS backbone) is also effective.The construction of such modified ODNs is described in detail in U.S.Pat. No. 6,900,187 B2, the contents of which are incorporated byreference. In some embodiments, the ODN is custirsen.

As used herein, “custirsen” refers to an antisense oligonucleotide thatreduces clusterin expression having the sequence CAGCAGCAGAGTCTTCATCAT(SEQ ID NO: 3), wherein the anti-clusterin oligonucleotide has aphosphorothioate backbone throughout, has sugar moieties of nucleotides1-4 and 18-21 bearing 2′-O-methoxyethyl modifications, has nucleotides5-17 which are 2′deoxynucleotides, and has 5-methylcytosines atnucleotides 1, 4, and 19. Custirsen is also known as TV-1011, OGX-011,ISIS 112989 and Custirsen Sodium.

As used herein, “RNA inducing molecule” refers to a molecule capable ofinducing RNA interference or “RNAi” of clusterin expression. RNAiinvolves mRNA degradation, but many of the biochemical mechanismsunderlying this interference are unknown. The use of RNAi has beendescribed in Fire et al., 1998, Carthew et al., 2001, and Elbashir etal., 2001, the contents of which are incorporated herein by reference.

Isolated RNA molecules can mediate RNAi. That is, the isolated RNAmolecules of the present invention mediate degradation or blockexpression of mRNA that is the transcriptional product of the gene,which is also referred to as a target gene. For convenience, such mRNAmay also be referred to herein as mRNA to be degraded. The terms RNA,RNA molecule(s), RNA segment(s) and RNA fragment(s) may be usedinterchangeably to refer to RNA that mediates RNA interference. Theseterms include double-stranded RNA, small interfering RNA (siRNA),hairpin RNA, single-stranded RNA, isolated RNA (partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA), aswell as altered RNA that differs from naturally occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the RNA or internally (at one or morenucleotides of the RNA). Nucleotides in the RNA molecules of the presentinvention can also comprise nonstandard nucleotides, includingnon-naturally occurring nucleotides or deoxyribonucleotides.Collectively, all such altered RNAi molecules are referred to as analogsor analogs of naturally-occurring RNA. RNA of the present invention needonly be sufficiently similar to natural RNA that it has the ability tomediate RNAi.

As used herein the phrase “mediate RNAi” refers to and indicates theability to distinguish which mRNA molecules are to be affected by theRNAi machinery or process. RNA that mediates RNAi interacts with theRNAi machinery such that it directs the machinery to 25 degradeparticular mRNAs or to otherwise reduce the expression of the targetprotein. In one embodiment, the present invention relates to RNAmolecules that direct cleavage of specific mRNA to which their sequencecorresponds. It is not necessary that there be perfect correspondence ofthe sequences, but the correspondence must be sufficient to enable theRNA to direct RNAi inhibition by cleavage or blocking expression of thetarget mRNA.

As noted above, the RNA molecules of the present invention in generalcomprise an RNA portion and some additional portion, for example adeoxyribonucleotide portion. The total number of nucleotides in the RNAmolecule is suitably less than in order to be effective mediators ofRNAi. In preferred RNA molecules, the number of nucleotides is 16 to 29,more preferably 18 to 23, and most preferably 21-23. Suitable sequencesare set forth in the present application as SEQ ID NOs:19 to 42 (Table2).

TABLE 2  Sequence Identification Numbers forRNA Interference Inducing Molecules SEQ ID NO: Sequence 19CCAGAGCUCG CCCUUCUACT T 20 GUAGAAGGGC GAGCUCUGGT T 21GAUGCUCAAC ACCUCCUCCT T 22 GGAGGAGGUG UUGAGCAUCT T 23UAAUUCAACA AAACUGUTT 24 GACAGUUUUA UUGAAUUAGT T 25 UAAUUCAACA AAACUGUTT26 ACAGUUUUGU UGAAUUATT 27 AUGAUGAAGA CUCUGCUGCT T 28GCAGCAGAGU CUUCAUCAUT T 29 UGAAUGAAGG GACUAACCUG TT 30CAGGUUAGUC CCUUCAUUCA TT 31 CAGAAAUAGA CAAAGUGGGG TT 32CCCCACUUUG UCUAUUUCUG TT 33 ACAGAGACUA AGGGACCAGA TT 34ACAGAGACUA AGGGACCAGA TT 35 CCAGAGCUCG CCCUUCUACT T 36GUAGAAGGGC GAGCUCUGGT T 37 GUCCCGCAUC GUCCGCAGCT T 38GCUGCGGACG AUGCGGGACT T 39 CUAAUUCAAU AAAACUGUCT T 40GACAGUUUUA UUGAAUUAGT T 41 AUGAUGAAGA CUCUGCUGC 42 GCAGCAGAGU CUUCAUCAU

The siRNA molecules of the invention are used in therapy to treatpatients, including human patients, that have cancers or other diseasesof a type where a therapeutic benefit is obtained by the inhibition ofexpression of the targeted protein. siRNA molecules of the invention areadministered to patients by one or more daily injections (intravenous,subcutaneous or intrathecal) or by continuous intravenous or intrathecaladministration for one or more treatment cycles to reach plasma andtissue concentrations suitable for the regulation of the targeted mRNAand protein.

As used herein, a “mammalian subject affected by prostate cancer” meansa mammalian subject who was been affirmatively diagnosed to haveprostate cancer.

As used herein, “androgen-independent prostate cancer” encompasses cellsand tumors containing cells that are not androgen-dependent (notandrogen sensitive); often such cells progress from beingandrogen-dependent to being androgen-independent. In some embodiments,androgen independent prostate cancer has progressed since theadministration of hormone ablation therapy and/or hormone blockadetherapy. In some embodiments, there is increased AR expression in theandrogen-independent prostate cancer compared to prostate cancer that isnot androgen-independent.

As used herein, “castration-resistant prostate cancer” encompasses anyandrogen-independent prostate cancer that is resistant to hormoneablation therapy and/or hormone blockade therapy. In some embodiments,castration-resistant prostate cancer has progressed since theadministration of hormone ablation or hormone blockade therapy. In someembodiments, there is increased AR expression in thecastration-resistant prostate cancer compared to prostate cancer that isnot castration resistant.

As used herein, “Hsp90 inhibitor” refers to an agent that perturbs orreduces a function of Hsp90, including inhibiting a Hsp90-proteininteraction, Hsp90 signaling, or Hsp90 protein expression. Hsp90inhibitors include but are not limited to Hsp90-specific monoclonalantibodies, oligonucleotides that target Hsp90 expression (such as Hsp90targeting antisense oligonucleotides or RNA inducing molecules), peptideagents specific for Hsp90, and small molecule inhibitors specific forHsp90. Non-limiting examples of Hsp90 inhibitors are Hsp90i-1, Hsp90i-2,Hsp90i-2-PRO and Hsp90i-2-PRO2.

Hsp90i-1 is a Hsp90 inhibitor. Hsp90i-1 is also known as17-allylamino-17-demethoxygeldanamycin (17-AAG), Telatinib,Tanespimycin, NSC-330507, CNF-101, KOS-953, GLD-36, and CP 127374. TheCAS Registry Number of Hsp90i-1 is 75747-14-7. The Hsp90i-1 used for theexperiments described herein is also referred to as 17-AAG and wasobtained from the National Institutes of Health (Bethesda, Md., USA).17-AAG has been discussed in Egorin et al., 1998, and Koga et al., 2009,and is also available for purchase from Invivogen (San Diego, Calif.,USA).

Hsp90i-2 is a Hsp90 inhibitor. Hsp90i-2 is also known as PF-04928473,and SNX-2112, and(4-(6,6-Dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-2-(4-hydroxy-cyclohexylamino)-benzamide).The CAS Registry No. for Hsp90i-2 is 908112-43-6. Hsp90i-2 has thefollowing structure:

Hsp90i-2 is discussed in Lamoureux et al., 2011, the entire contents ofwhich are incorporated herein by reference.

Hsp90i-2-PRO is a Hsp90 inhibitor. Hsp90i-2-PRO is the prodrug ofHsp90i-2. The CAS Registry No. for Hsp90i-2-PRO is 908115-27-5.Hsp90i-2-PRO is also known as SNX-5422 and PF-04929113. Hsp90i-2-PRO hasthe following structure:

Hsp90i-2-PRO is discussed in Lamoureux et al., 2011, the entire contentsof which are incorporated herein by reference.

Hsp90i-2-PRO2 is another prodrug of Hsp90i-2. Hsp90i-2-PRO2 is discussedin Chandarlapaty et al., 2008, the entire contents of which areincorporated herein by reference. Hsp90i-2-PRO2 is also known asSNX-5542.

Methods of synthesis for Hsp90i-2, Hsp90i-2-PRO and Hsp90i-2-PRO2 aredescribed in Huang et al., J. Med Chem. 52:4288-4305 (2009), and U.S.Pat. No. 7,928,135, the entire contents of which are incorporated hereinby reference. Alternatively, Hsp90i-2, Hsp90i-2-PRO and Hsp90i-2-PRO2are available from Pfizer Inc. (New York, N.Y., USA) and Serenex Inc.(Durham, N.C., USA).

Those having ordinary skill in the art of organic synthesis willappreciate that modifications to the general procedures shown in thesynthesis schemes of this application can be made to yield structurallydiverse compounds. For example, where aryl rings are present, allpositional isomers are contemplated and may be synthesized usingstandard aromatic substitution chemistry. The number and types ofsubstituents may also vary around the aryl rings. Furthermore, wherealkyl groups are present, the chain length may be modified using methodswell known to those of ordinary skill in the art. Where ester formationis contemplated, lactones may be used wherein the lactone ring is openedby reaction with a nucleophile, such as an ether-containing moietydescribed hereinabove. Suitable organic transformations are described inMarch's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure(Wiley-Interscience; 6th edition, 2007), the content of which is herebyincorporated by reference.

Compounds of the subject invention can be converted to prodrugs tooptimize absorption and bioavailability. Formation of a prodrug include,but is not limited to, reaction of a free hydroxyl group with acarboxylic acid to form an ester, reaction of a free hydroxyl group withan phosphorus oxychloride followed by hydrolysis to form a phosphate, orreaction of a free hydroxyl group with an amino acid to form an aminoacid ester, the process of which has been described previously byChandran in WO 2005/046575. The substituents are chosen and resultinganalogs are evaluated according to principles well known in the art ofmedicinal and pharmaceutical chemistry, such as quantification ofstructure-activity relationships, optimization of biological activityand ADMET (absorption, distribution, metabolism, excretion, andtoxicity) properties.

Except where otherwise specified, when the structure of a compound ofthis invention includes an asymmetric carbon atom, it is understood thatthe compound occurs as a racemate, racemic mixture, and isolated singleenantiomer. All such isomeric forms of these compounds are expresslyincluded in this invention. Except where otherwise specified, eachstereogenic carbon may be of the R or S configuration. It is to beunderstood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis, such as those described in“Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S.Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolutionmay be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atomsoccurring on the compounds disclosed herein. Isotopes include thoseatoms having the same atomic number but different mass numbers. By wayof general example and without limitation, isotopes of hydrogen includetritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughoutthis application, when used without further notation, are intended torepresent all isotopes of carbon, such as ¹²C, ¹³C, or ¹⁴C. Furthermore,any compounds containing ¹³C or ¹⁴C may specifically have the structureof any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structuresthroughout this application, when used without further notation, areintended to represent all isotopes of hydrogen, such as ¹H, ²H, or ³H.Furthermore, any compounds containing ²H or ³H may specifically have thestructure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventionaltechniques known to those skilled in the art using appropriateisotopically-labeled reagents in place of the non-labeled reagentsemployed.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C₁-C_(n) asin “C₁-C_(n) alkyl” is defined to include groups having 1, 2, . . . ,n−1 or n carbons in a linear or branched arrangement. For example,C₁-C₆, as in “C₁-C₆ alkyl” is defined to include groups having 1, 2, 3,4, 5, or 6 carbons in a linear or branched arrangement, and specificallyincludes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl,hexyl, and octyl.

As used herein, “aryl” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring,wherein at least one ring is aromatic, and may be unsubstituted orsubstituted. Examples of such aryl elements include phenyl, p-toluenyl(4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, phenanthryl,anthryl or acenaphthyl. In cases where the aryl substituent is bicyclicand one ring is non-aromatic, it is understood that attachment is viathe aromatic ring.

The term “heteroaryl”, as used herein, represents a stable monocyclic,bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein atleast one ring is aromatic and contains from 1 to 4 heteroatoms selectedfrom the group consisting of O, N and S. Bicyclic aromatic heteroarylgroups include but are not limited to phenyl, pyridine, pyrimidine orpyridizine rings that are (a) fused to a 6-membered aromatic(unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a5- or 6-membered aromatic (unsaturated) heterocyclic ring having twonitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated)heterocyclic ring having one nitrogen atom together with either oneoxygen or one sulfur atom; or (d) fused to a 5-membered aromatic(unsaturated) heterocyclic ring having one heteroatom selected from O, Nor S. Heteroaryl groups within the scope of this definition include butare not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl,indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl,isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl,oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl,thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl,1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl,dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

The alkyl, aryl, and heteroaryl substituents may be substituted orunsubstituted, unless specifically defined otherwise.

The compounds of the instant invention may be in a salt form. As usedherein, a “salt” is a salt of the instant compounds which has beenmodified by making acid or base salts of the compounds. In someembodiments, the salt is pharmaceutically acceptable. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as phenols. The salts can bemade using an organic or inorganic acid. Such acid salts are chlorides,bromides, sulfates, nitrates, phosphates, sulfonates, formates,tartrates, maleates, malates, citrates, benzoates, salicylates,ascorbates, and the like. Phenolate salts are the alkaline earth metalsalts, sodium, potassium or lithium. The term “pharmaceuticallyacceptable salt” in this respect, refers to the relatively non-toxic,inorganic and organic acid or base addition salts of compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, or byseparately reacting a purified compound of the invention in its freebase or free acid form with a suitable organic or inorganic acid orbase, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The compositions of this invention may be administered in various forms,including those detailed herein. The treatment with the compound may bea component of a combination therapy or an adjunct therapy, i.e. thesubject or patient in need of the drug is treated or given another drugfor the disease in conjunction with one or more of the instantcompounds. This combination therapy can be sequential therapy where thepatient is treated first with one drug and then the other or the twodrugs are given simultaneously. These can be administered independentlyby the same route or by two or more different routes of administrationdepending on the dosage forms employed.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable solvent, suspending agent or vehicle, fordelivering the instant compounds to the animal or human. The carrier maybe liquid or solid and is selected with the planned manner ofadministration in mind. Liposomes are also a pharmaceutically acceptablecarrier.

The dosage of the compounds administered in treatment will varydepending upon factors such as the pharmacodynamic characteristics of aspecific chemotherapeutic agent and its mode and route ofadministration; the age, sex, metabolic rate, absorptive efficiency,health and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment being administered; thefrequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds may comprise a single compound ormixtures thereof with additional anticancer agents. The compounds can beadministered in oral dosage forms as tablets, capsules, pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions. Thecompounds may also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, or introduceddirectly, e.g. by injection, topical application, or other methods, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts.

The compounds can be administered in admixture with suitablepharmaceutical diluents, extenders, excipients, or carriers(collectively referred to herein as a pharmaceutically acceptablecarrier) suitably selected with respect to the intended form ofadministration and as consistent with conventional pharmaceuticalpractices. The unit will be in a form suitable for oral, rectal,topical, intravenous or direct injection or parenteral administration.The compounds can be administered alone or mixed with a pharmaceuticallyacceptable carrier. This carrier can be a solid or liquid, and the typeof carrier is generally chosen based on the type of administration beingused. The active agent can be co-administered in the form of a tablet orcapsule, liposome, as an agglomerated powder or in a liquid form.Examples of suitable solid carriers include lactose, sucrose, gelatinand agar. Capsule or tablets can be easily formulated and can be madeeasy to swallow or chew; other solid forms include granules, and bulkpowders. Tablets may contain suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Examples of suitable liquid dosage formsinclude solutions or suspensions in water, pharmaceutically acceptablefats and oils, alcohols or other organic solvents, including esters,emulsions, syrups or elixirs, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules and effervescentpreparations reconstituted from effervescent granules. Such liquiddosage forms may contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents. Oral dosage forms optionally contain flavorants andcoloring agents. Parenteral and intravenous forms may also includeminerals and other materials to make them compatible with the type ofinjection or delivery system chosen.

Techniques and compositions for making dosage forms useful in thepresent invention are described in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents,coloring agents, flavoring agents, flow-inducing agents, and meltingagents. For instance, for oral administration in the dosage unit form ofa tablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol,sorbitol and the like. Suitable binders include starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamallar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine, orphosphatidylcholines. The compounds may be administered as components oftissue-targeted emulsions.

The compounds may also be coupled to soluble polymers as targetable drugcarriers or as a prodrug. Such polymers include polyvinylpyrrolidone,pyran copolymer, polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compounds andpowdered carriers, such as lactose, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as immediate release products or as sustained releaseproducts to provide for continuous release of medication over a periodof hours. Compressed tablets can be sugar coated or film coated to maskany unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration in the gastrointestinaltract.

For oral administration in liquid dosage form, the oral drug componentsare combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Examples ofsuitable liquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, a suitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The compounds of the instant invention may also be administered inintranasal form via use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration willgenerally be continuous rather than intermittent throughout the dosageregimen.

Parenteral and intravenous forms may also include minerals and othermaterials to make them compatible with the type of injection or deliverysystem chosen.

The inhibition of clusterin expression may be transient, and may occurin combination with a Hsp90 inhibitor. In humans with prostate cancer,this means that inhibition of expression should be effective startingwithin a day or two of Hsp90 inhibition or administration of an Hsp90inhibitor, and extending for about 3 to 6 months thereafter. This mayrequire multiple doses to accomplish. It will be appreciated, however,that the period of time may be more prolonged, starting Hsp90 inhibitionand extending for substantial time afterwards without departing from thescope of the invention.

Aspects of the invention can be applied to the treatment ofandrogen-independent prostate cancer, or to prevent prostate cancer frombecoming androgen-independent.

Aspects of the invention can be applied to the treatment ofcastration-resistant prostate cancer, or to prevent prostate cancer frombecoming castration-resistant.

“Combination” means either at the same time and frequency, or moreusually, at different times and frequencies as an oligonucleotidetargeting clusterin expression, as part of a single treatment plan.Aspects of the invention include the administration of theoligonucleotide before, after, and/or during the administration of aHsp90 inhibitor. A Hsp90 inhibitor may therefore be used, in combinationwith an oligonucleotide according to the invention, but yet beadministered at different times, different dosages, and at a differentfrequency, than the oligonucleotide.

As used herein, an “amount” or “dose” of an oligonucleotide measured inmilligrams refers to the milligrams of oligonucleotide present in a drugproduct, regardless of the form of the drug product.

As used herein, “effective” when referring to an amount ofoligonucleotide which reduces clusterin expression, a Hsp90 inhibitor,or any combination thereof refers to the quantity of oligonucleotide,Hsp90 inhibitor, or any combination thereof that is sufficient to yielda desired therapeutic response without undue adverse side effects (suchas toxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.

As used herein, “treating” encompasses, e.g., inhibition, regression, orstasis of the progression of prostate cancer. Treating also encompassesthe prevention or amelioration of any symptom or symptoms of prostatecancer.

As used herein, “inhibition” of disease progression or diseasecomplication in a subject means preventing or reducing the diseaseprogression and/or disease complication in the subject.

As used herein, a “symptom” associated with prostate cancer includes anyclinical or laboratory manifestation associated with prostate cancer,and is not limited to what the subject can feel or observe.

As used herein, “pharmaceutically acceptable carrier” refers to acarrier or excipient that is suitable for use with humans and/or animalswithout undue adverse side effects (such as toxicity, irritation, andallergic response) commensurate with a reasonable benefit/risk ratio. Itcan be a pharmaceutically acceptable solvent, suspending agent orvehicle, for delivering the instant compounds and/or combinations to thesubject.

The Following Abbreviations are Used Herein

PCa prostate cancer

CRPC castrate resistant prostate cancer

HSP heat shock proteins

CLU clusterin

PSA prostate specific antigen

17-AAG 17-allylamino-17-demethoxygeldanamycin

Aso antisense oligonucleotide

Dosage Units

Administration of an oligonucleotide that targets clusterin expressioncan be carried out using the various mechanisms known in the art,including naked administration and administration in pharmaceuticallyacceptable lipid carriers. For example, lipid carriers for antisensedelivery are disclosed in U.S. Pat. Nos. 5,855,911 and 5,417,978, whichare incorporated herein by reference. In general, the oligonucleotide isadministered by intravenous (i.v.), intraperitoneal (i.p.), subcutaneous(s.c.), or oral routes, or direct local tumor injection. In preferredembodiments, an oligonucleotide targeting clusterin expression isadministered by i.v. injection. In some embodiments, the amount ofoligonucleotide administered is 640 mg.

The amount of antisense oligonucleotide administered is one effective toinhibit the expression of clusterin in prostate cells. It will beappreciated that this amount will vary both with the effectiveness ofthe antisense oligonucleotide employed, and with the nature of anycarrier used.

The amount of antisense oligonucleotide targeting clusterin expressionadministered may be from 40 to 640 mg, or 300-640 mg. Administration ofthe antisense oligonucleotide may be once in a seven day period, 3 timesa week, or more specifically on days 1, 3 and 5, or 3, 5 and 7 of aseven day period. In some embodiments administration of the antisenseoligonucleotide is less frequent than once in a seven day period.Dosages may be calculated by patient weight, and therefore a dose rangeof about 1-20 mg/kg, or about 2-10 mg/kg, or about 3-7 mg/kg, or about3-4 mg/kg could be used. This dosage is repeated at intervals as needed.One clinical concept is dosing once per week with 3 loading doses duringweek one of treatment. The amount of antisense oligonucleotideadministered is one that has been demonstrated to be effective in humanpatients to inhibit the expression of clusterin in cancer cells.

In some embodiments of the invention, the amount of oligonucleotidetargeting the expression of clusterin required for treatment of prostatecancer is less in combination with a Hsp90 inhibitor, than would berequired with oligonucleotide monotherapy.

Custirsen may be formulated at a concentration of 20 mg/mL as anisotonic, phosphate-buffered saline solution for IV administration andcan be supplied as an 8 mL solution containing 160 mg custirsen sodiumin a single vial.

Custirsen may be added to 250 mL 0.9% sodium chloride (normal saline).The dose may be administered using either a peripheral or centralindwelling catheter intravenously as an infusion over 2 hours.Additionally, an infusion pump may be used.

Administration of an Hsp90 inhibitor may be oral, nasal, pulmonary,parenteral, i.v., i.p., intra-articular, transdermal, intradermal, s.c.,topical, intramuscular, rectal, intrathecal, intraocular, and buccal.One of skill in the art will recognize that higher doses may be requiredfor oral administration than for i.v. injection.

The dose of Hsp90 inhibitor may be 60 mg/kg, 55 mg/kg, 45 mg/kg, 40mg/kg, 35 mg/kg, 25 mg/kg, 20 mg/kg, 15 mg/kg, 10 mg/kg, 5 mg/kg orless.

A dosage unit of the oligonucleotide which reduces clusterin expressionand an Hsp90 inhibitor may comprise one of each singly or mixturesthereof. A combination of an oligonucleotide which reduces clusterinexpression and an Hsp90 inhibitor can be administered in oral dosageforms as tablets, capsules, pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. An oligonucleotide whichreduces clusterin expression and/or Hsp90 inhibitor may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, or introduced directly, e.g. byinjection or other methods, into or onto a prostate cancer lesion, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts.

An oligonucleotide which reduces clusterin expression and/or Hsp90inhibitor can be administered in admixture with suitable pharmaceuticaldiluents, extenders, excipients, or carriers (collectively referred toherein as a pharmaceutically acceptable carrier) suitably selected withrespect to the intended form of administration and as consistent withconventional pharmaceutical practices. The unit will be in a formsuitable for oral, rectal, topical, intravenous or direct injection orparenteral administration. The oligonucleotide and/or Hsp90 inhibitorcan be administered alone or mixed with a pharmaceutically acceptablecarrier. This carrier can be a solid or liquid, and the type of carrieris generally chosen based on the type of administration being used.Capsule or tablets can be easily formulated and can be made easy toswallow or chew; other solid forms include granules, and bulk powders.Tablets may contain suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Examples of suitable liquid dosage formsinclude solutions or suspensions in water, pharmaceutically acceptablefats and oils, alcohols or other organic solvents, including esters,emulsions, syrups or elixirs, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules and effervescentpreparations reconstituted from effervescent granules. Such liquiddosage forms may contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents. Oral dosage forms optionally contain flavorants andcoloring agents. Parenteral and intravenous forms may also includeminerals and other materials to make them compatible with the type ofinjection or delivery system chosen.

An oligonucleotide which reduces clusterin expression and/or Hsp90inhibitor can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamallar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine, orphosphatidylcholines. The compounds may be administered as components oftissue-targeted emulsions.

For oral administration in liquid dosage form, an Hsp90 inhibitor may becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Examples ofsuitable liquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents.

In some embodiments of the invention, the amount of Hsp90 inhibitorrequired for treatment of prostate cancer is less in combination with anoligonucleotide targeting the expression of clusterin, than would berequired with Hsp90 monotherapy.

A dosage unit may comprise a single compound or mixtures of compounds. Adosage unit can be prepared for oral or injection dosage forms.

According to an aspect of the invention, there is provided anoligonucleotide which reduces clusterin expression-containingpharmaceutical composition packaged in dosage unit form, wherein theamount of the oligonucleotide in each dosage unit is 640 mg or less.Said pharmaceutical composition may include an Hsp90 inhibitor, and maybe in an injectable solution or suspension, which may further containsodium ions.

According to another aspect of the invention, there is provided the useof an oligonucleotide targeting clusterin expression and a Hsp90inhibitor in the manufacture of a medicament for the treatment ofcancer, where the medicament is formulated to deliver a dosage of 640 mgor less of oligonucleotide to a patient. The medicament may containsodium ions, and/or be in the form of an injectable solution.

General techniques and compositions for making dosage forms useful inthe present invention are described in the following references: 7Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors,1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981);Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modern Pharmaceutics Drugs and thePharmaceutical Sciences, Vol. 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). These references in their entireties are herebyincorporated by reference into this application.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS Example 1. Hsp90 Inhibitors Induce Expression ofBSPs in Prostate Cancer (PCa) Cells In Vitro and In Vivo

Dose- and time-dependent effects of Hsp90i-1 or Hsp90i-2 on theexpression of CLU, Hsp90, Hsp70 and Akt protein and mRNA levels wasevaluated in LNCaP and PC-3 cells. Both Hsp90i-1 and Hsp90i-2 increasedHsp70 and CLU protein levels up to 3 fold in a dose- and time-dependentmanner (FIGS. 1A, B and C). Hsp90 inhibition induced a dose- and a timedependent decline of Akt expression as previously reported (Lamoureux etal., 2011). mRNA levels of CLU, Hsp70 and Hsp90 also increased afterHsp90 inhibitor treatment (FIG. 1D).

Next the effects of Hsp90i-2 treatment on CLU expression were assessedin vivo in CRPC LNCaP xenografts using immunohistochemistry and westernblot (FIG. 2). CLU expression increased 4-fold after treatment withHsp90i-2-PRO (***, p<0.001) compared with vehicle treated tumor (FIG.2A, B). Similarly, Hsp70, which is considered a pharmacodynamic measureof Hsp90 inhibition (Solit et al., 2003; Eccles et al., 2008), increased2.3-fold after treatment with Hsp90i-2-PRO (***, p<0.001) (FIG. 2A).

Example 2. Treatment-Induced Feed Forward Loop Involving CLU and HSF-1Activity

Since HSF-1 is the pre-dominant regulator of the heat shock response(Banerji et al., 2008; Workman et al., 2007), the effect of Hsp90inhibition on HSF-1-activity and expression of HSPs was evaluated.Hsp90i-1 or Hsp90i-2 significantly induced CLU (FIG. 1) as well as HSF-1activity in a dose-dependent manner (***, p≦0.001; FIG. 3A). CLUoverexpression protected PC-3 tumor cells from Hsp90i-2-inducedapoptosis (**, p≦0.01; FIG. 8A). Moreover, HSF-1 knockdown using siRNAdecreases CLU expression, sensitizing tumor cells to apoptosis-inducedby Hsp90i-2 (FIG. 8B), confirming that the protective effect of CLU ismediated by HSF-1. Surprisingly, overexpression of CLU also increasedHSF-1 activity (***, p≦0.001, FIG. 3B), while CLU knockdown using siRNAor custirsen significantly decreased HSF-1 activity (*, p≦0.05; ***,p≦0.001; FIG. 3C), identifying novel feed-forward regulation of HSF-1 byCLU. Indeed, silencing of CLU inhibited HSF-1 transcriptionalactivity-induced by Hsp90i-1 or Hsp90i-2 (FIG. 3C), as well as HSF-1regulated genes such as Hsp27 and Hsp70 (FIG. 3D). This effect can beexplained by the ability of CLU knockdown to sequester HSF-1 in thecytoplasm (FIG. 8B).

Example 3. Increased Potency of Combination Treatment ComprisingCustirsen and Hsp90 Inhibitor in Increasing Apoptosis in Prostate TumorCell Lines Compared to Monotherapy

Since Hsp90 inhibitors induce up-regulation of CLU and functions as amediator in treatment resistance (Zoubeidi et al. 2010; Gleave et al.,2005; Zellweger et al., 2003), it was next evaluated if CLU knockdowncombined with Hsp90 inhibition increased treatment effectiveness. LNCaPcells were treated with custirsen and subsequently treated withindicated concentrations of Hsp90i-1 or Hsp90i-2. The combination hadsignificantly enhanced Hsp90i-1 or Hsp90i-2 effectiveness, reducing cellviability by 20% at 100 nM and 1000 nM (*, p<0.05) compared with cellstreated with control ScrB ASO and Hsp90 inhibitors (FIG. 4A). Todetermine whether this effect was additive or a combination effect, thedose-dependent effects

with constant ratio design and the combination index (CI) values wasperformed and calculated according to the Chou and Talalay median effectprincipal (Chou et al., 1984). FIG. 4B shows the dose response curve(combination treatment, custirsen or HSP90i-2 monotherapy) and thecombination index plots, indicating that custirsen and HSP90i-2 hadenhanced combined potency on tumor cell growth compared to custirsen orHsp90i-2 inhibitor monotherapy.

Moreover, OGX-011 potentiates the effect of Hsp90 inhibitor to induceapoptosis (FIGS. 4C and D). Flow cytometric analysis shows thatapoptotic rates (subG1 fraction) increased significantly (p<0.001) whencustirsen is combined with Hsp90i-1 (53%) or Hsp90i-2(65.4%), comparedto control ScrB ASO (4.2%), custirsen alone (17.4%), control ScrBASO+Hsp90i-1 (18.3%) or control ScrB ASO+Hsp90i-2 (24.8%; FIG. 4C).Moreover, the combination custirsen with Hsp90i-1 or Hsp90i-2 inducedmore caspase-dependent apoptosis compared to Hsp90 inhibitor- orcustirsen monotherapy, as shown by cleaved PARP and caspase-3 expression(FIG. 4C). The significant increase of caspase-3 activity confirms thatcustirsen sensitizes cells to Hsp90 inhibition with increased apoptoticrates (FIG. 4D). Reduced cell viability from combined CLU plus Hsp90inhibition results, in part, from decreases in p-Akt levels in both PC-3and LNCaP cells, as well as AR (and PSA) expression in LNCaP cells (FIG.4C).

Example 4. Potent Combination Therapy of Custirsen and Hsp90i-1 in PC-3Xenografts In Vivo

The effects of combining custirsen with Hsp90i-1 was evaluated in PC-3tumors in vivo. Male nude mice bearing PC-3 xenografts were randomlyselected for treatment (custirsen+Hsp90i-1 vs control ScrB+Hsp90i-1;n-7). Custirsen+Hsp90i-1 had significantly enhanced the antitumoreffects compared to of ScrB+Hsp90i-1 in vivo, reducing the mean tumorvolume from 2935.3 mm³ to 1176.9 mm³ after 68 days (**; p≦0.01),compared to control ScrB (FIG. 5A). Cancer specific survival wassignificantly prolonged with combined custirsen+Hsp90i-1 compared withcontrols (71.4% vs 14.3% at day 72, respectively; *; p≦0.05; FIG. 5B.Immunohistochemical analysis reveals decreased CLU, Ki67, and Akt,expression after treatment with custirsen+Hsp90i-1 compared to othergroups (FIG. 5C). Additionally, custirsen+Hsp90i-1 treated tumors hadhigher apoptosis as shown by increased TUNEL staining compared withother groups (FIG. 5C).

Example 5. Potent Combination Therapy of Custirsen and Hsp90i-2-PRO inLNCaP CRPC Zenografts In Vivo

Next the effects of combined treatment with custirsen and Hsp90i-2-PROwas assessed in castrate resistant LNCaP tumors. Mice bearing LNCaPtumors were castrated when PSA values exceeded 50 ng/ml. Once PSA levelsrelapsed above pre-castration levels mice were randomly assigned tovehicle control, Hsp90i-2-PRO alone, Hsp90i-2-PRO+control ScrB, orHsp90i-2-PRO+custirsen (n-10 in each group). Mice treated withHsp90i-2-PRO+custirsen had significant delays in tumor growth comparedwith all other groups (FIG. 6A) (at 10 days, respectively 265.3 mm³, and892.7 mm³ for control, 646.4 mm³ for Hsp90i-2-PRO alone and 551.56 mm³for Hsp90i-2-PRO+control ScrB). By 7 weeks post treatment, all mice inthe control had been euthanized; tumor volume in theHsp90i-2-PRO+custirsen group was r517.4 mm³ compared to 2483.6 mm3 forHSP90i-2-PRO alone and 2176.4 mm³ for Hsp90i-2-PRO+control ScrB; ***,p<0.001; FIG. 6A).

Serum PSA levels were also significantly lower (˜4-fold) in the micereceiving custirsen+Hsp90i-2-PRO compared with other groups (***,p<0.001; FIG. 6B). The combination custirsen+Hsp90i-2-PRO group had amean PSA level of 120 ng/ml after 42 days compared to 418.7 ng/ml invehicle group, 527 ng/ml in Hsp90i-2-PRO alone, or 480.3 ng/ml inscrB+Hsp90i-2-PRO groups. The combination custirsen+Hsp90i-2-PRO grouphad a significantly increased PSA doubling time (33.6 weeks; *, p<0.05)and decreased PSA velocity (13.78 ng/mL/week; *, p<0.05) compared withother groups (PSA doubling time: ˜2.4 weeks; velocity: ˜85 ng/mL/week;FIG. 6C).

Overall survival was also significantly longer in mice treated withcombined custirsen+Hsp90i-2-PRO (FIG. 6D). By day 57, all mice died orwere euthanized due to high tumor burden in control, Hsp90i-2-PRO alone,or control ScrB+Hsp90i-2-PRO groups compared with the combinedcustirsen+Hsp90i-2-PRO group, where all mice were still alive (p<0.001)after 62 days. These data demonstrate that targeting CLU using custirsenin combination with HSP90i-2-PRO inhibits tumor growth and prolongssurvival in human CRPC xenograft model significantly more thanmonotherapy.

Consistent with in vitro findings, immunohistochemical analysis revealeddecreased CLU, Ki67, Akt, and AR expression after treatment withcombined custirsen+Hsp90i-2-PRO compared with other groups (FIG. 7A).The immunostaining results were corroborated by western blotting (FIG.7B). Additionally, tumors treated with combinationcustirsen+Hsp90i-2-PRO had higher apoptosis rates compared with othergroups as shown by increased TUNEL staining (FIG. 7A). These datasuggest that decreases in tumor progression custirsen+Hsp90i-2-PROtreated tumors result from both reduced proliferation rates as well asincreased apoptosis rates.

Example 6. Materials and Methods for Examples 1-5

Tumor Cell Lines and Reagents:

The human PCa cell line PC-3 was purchased from the American TypeCulture Collection (2008, ATCC-authentication by isoenzymes analysis)and maintained in DMEM (Invitrogen-Life Technologies, Inc.) supplementedwith 5% fetal bovine serum and 2 mmol/L Lglutamine. LNCaP cells werekindly provided by Dr. Leland W. K. Chung (1992, MDACC, Houston Tx) andtested and authenticated by whole-genome and whole-transcriptomesequencing on Illumina Genome Analyzer IIx platform in July 2009. LNCaPcells were maintained RPMI 1640 (Invitrogen Life Technologies, Inc.)supplemented with 5% fetal bovine serum and 2 mmol/L L-glutamine. Allcell lines were cultured in a humidified 5% CO₂/air atmosphere at 37° C.All cell lines were passaged for less than 3 months after resurrection.Western blotting and/or real time PCR was performed for AR and PSA eachtime when LNCaP cells were resurrected.

Therapeutic Agents:

Hsp90 inhibitor, HSP90i-2(4-(6,6-Dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-2-(4-hydroxy-cyclohexylamino)-benzamide)and its prodrug HSP90i-2-PRO were used respectively for in vitro and invivo studies. These compounds are novel synthetic small molecular weightinhibitors that bind the N-terminal adenosine triphosphate binding siteof Hsp90 and HSP90i-2-PRO is orally bioavailable. For the in vitrostudies, HSP90i-2 was dissolved in dimethyl sulfoxide (DMSO) at 10 mMstock solutions and stored at −20° C. For the in vivo studies,HSP90i-2-PRO was dissolved in PBS 1% carboxymethylcellulose and 0.5%Tween 80 (Invitrogen-Life Technologies, Inc.) at 15 mg/ml and stored at4° C.

HSP90i-1 (17-allylamino-17-demethoxygeldanamycin (17-AAG)) was used forin vitro and in vivo studies. For the studies, 17-AAG was dissolved indimethyl sulfoxide (DMSO) at 10 mM stock solutions and stored at −20° C.

Clusterin siRNA and Antisense Oligonucleotides

siRNAs were purchased from Dharmacon Research, Inc. (Lafayette, Colo.)using the siRNA sequence corresponding to the human CLU initiation sitein exon 2 and a scramble control as previously described (Sowery et al.,2008). Second-generation antisense (custirsen) and scrambled (ScrB)oligonucleotides with a 2′-O-(2-methoxy)ethyl modification were suppliedby OncoGenex Pharmaceuticals (Vancouver, British Columbia, Canada).Custirsen sequence (5′-CAGCAGCAGAGTCTTCATCAT-3′), SEQ ID NO:3corresponds to the initiation site in exon II of human CLU. The ScrBcontrol sequence was 5′-CAGCGCTGACAACAGTTTCAT-3′ (SEQ ID NO: 44).Prostate cells were treated with siRNA or oligonucleotides usingprotocols described previously (Sowery et al., 2008).

Cell Proliferation and Apoptosis Assays:

Prostate cells lines were plated in appropriate media (DMEM or RPMI)with 5% FBS and treated with Hsp90i-2-PRO or Hsp90i-1 at indicatedconcentration and time and cell growth was measured using the crystalviolet assay as described previously (Leung et al., 2000). Detection andquantitation of apoptotic cells were done by flow-cytometry (describedbelow) and western blotting analysis. Each assay was repeated intriplicate.

The combination index (CI) was evaluated using CalcuSyn dose effectanalysis software (Biosoft, Cambridge, UK). This method, based on themultiple drug effect equation of Chou-Talalay (Chou et al., 1984), issuitable for calculating combined drug activity over a wide range ofgrowth inhibition: CI=1, additivity; CI>1, antagonism; CI<1, combinationeffect. CI was calculated at ED₅₀ and ED₇₅.

Caspase-3 activity was assessed 3 days after treatment using the kitCaspACE Assay System, Fluorometric (Promega, Madison, Wis., USA). Fiftyμg of total cell lysate were incubated with caspase-3 substrateAC-DEVD-AMC at room temperature for 4 h and caspase-3 activity wasquantified in a fluorometer with excitation at 360 nm and emission 460nm.

Cell Cycle Analysis:

Prostate cancer cell lines were incubated in the absence or the presenceof 1 μM Hsp90i-2 or Hsp90i-1 for 72 h, trypsinized, washed twice andincubated in PBS containing 0.12% Triton X-100, 0.12 mM EDTA and 100μg/ml ribonuclease A; 50 μg/ml propidium iodide was then added to eachsample for 20 min at 4° C. Cell cycle distribution was analyzed by flowcytometry (Beckman Coulter Epics Elite, Beckman, Inc., Miami, Fla.),based on 2N and 4N DNA content. Each assay was done in triplicate.

Western Blotting Analysis:

Samples containing equal amounts of protein (depending on the antibody,5-50 μg) from lysates of cultured tumor prostate cell lines underwentelectrophoresis on SDS-polyacrylamide gel and were transferred tonitrocellulose filters. The filters were blocked in Odyssey BlockingBuffer (LI-COR Biosciences) at room temperature for 1 h and blots wereprobed overnight at 4° C. with primary antibodies to detect proteins ofinterests. After incubation, the filters were washed 3 times withwashing buffer (PBS containing 0.1% Tween) for 5 min. Filters were thenincubated for 1 h with 1:5,000 diluted Alexa Fluor secondary antibodies(Invitrogen) at room temperature. Specific proteins were detected usingODYSSEY IR imaging system (LI-COR Biosciences) after washing.

Quantitative Reverse Transcription-PCR:

Total RNA was extracted from cultured cells after 48 h of treatmentusing TRIzol reagent (Invitrogen Life Technologies, Inc.). Two μg oftotal RNA was reversed transcribed using the Transcriptor First StrandcDNA Synthesis Kit (Roche Applied Science). Real-time monitoring of PCRamplification of complementary DNA (cDNA) was performed using DNAprimers (supplemental table Si) on ABI PRISM 7900 HT Sequence DetectionSystem (applied Biosystems) with SYBR PCR Master Mix (AppliedBiosystems). Target gene expression was normalized to GAPDH levels inrespective samples as an internal standard, and the comparative cyclethreshold (Ct) method was used to calculated relative quantification oftarget mRNAs. Each assay was performed in triplicate.

Luciferase Assay:

LNCaP and C4-2 cells (2.5×10⁵) were plated on six-plates and transfectedusing lipofectin (6 μL per well; Invitrogen Life Technologies, Inc.).The total amount HSE plasmids DNA used were normalized to 1 μg per wellby the addition of a control plasmid. One μM HSP90i-2 or Hsp90i-1 wasadded 4 h after the transfection and for 48 h. HSE-luciferase activitywas measured using Dual-Luciferase Reporter Assay System (Promega) withthe aid of a microplate luminometer (EG&G Berthold). All experimentswere carried out in triplicate wells and repeated 3 times usingdifferent preparations of plasmids.

Immunofluorescence:

Tumor cells were grown on coverslips and treated with differentconcentration of Hsp90i-2 or Hsp90i-1 for 48 h. After treatment, cellswere fixed in ice-cold methanol completed with 3% acetone for 10 min at−20° C. Cells were the washed thrice with PBS and incubated with 0.2%Triton/PBS for 10 min, followed by washing and 30 min blocking in 3%nonfat milk before the addition of antibody overnight to detect HSF-1(1:250). Antigens were visualized using anti-mouse antibody coupled withFITC (1:500; 30 min). Photomicrographs were taken at 20× magnificationusing Zeiss Axioplan II fluorescence microscope, followed by analysiswith imaging software (Northern Eclipse, Empix Imaging, Inc.).

Animal Treatment:

Male athymic nude mice (Harlan Sprague-Dawley, Inc.) were injected s.c.with 2×10⁶ LNCaP cells (suspended in 0.1 mL Matrigel; BD Biosciences).The mice were castrated once tumors reach between 300 and 500 mm³ or thePSA level increased above 50 ng/mL. Once tumors progressed to castrateresistance, mice were randomly assigned to vehicle, Hsp90i-2-PRO alone,Hsp90i-2-PRO+ScrB ASO or Hsp90i-2-PRO+custirsen. Hsp90i-2-PRO (Prodrug,25 mg/kg; formulation in 0.5% CMC+0.5% Tween-80) is orally administeredthree times per week and custirsen or ScrB ASO (15 mg/kg) was injectedintra-peritoneally once daily for the first week and then three timesper week. Each experimental group consisted of 10 mice. Tumor volume wasmeasured twice weekly (length×width×depth×0.5432). Serum PSA wasdetermined weekly by enzymatic immunoassay (Abbott IMX, Montreal,Quebec, Canada). PSA doubling time (PSAdt) and velocity were calculatedby the log-slope method (PSAt=PSAinitial×emt). Data points wereexpressed as average tumor volume±SEM or average PSA concentration±SEM.

To establish PC-3 tumors, 2×10^(b) PC-3 cells were inoculated s.c. inthe flank region of 6-8 week-old male athymic mice (HarlanSprague-Dawley, Inc.). When tumors reached 100 mm³, usually 3-4 weeksafter injection, mice were randomly selected for treatment with Hsp90i-1(25 mg/kg)+control ScrB ASO (15 mg/kg) or Hsp90i-1+custirsen (15 mg/kg).Hsp90i-1 was injected i.p. three times per week, and custirsen or ScrBwere injected i.p. once/day for the first week and then three times perweek. For each experimental group consisted of 7 mice. Tumor volume wasmeasured twice weekly. Data points were expressed as average tumorvolume±SEM.

When tumor volume reached 210% of body weight, mice were sacrificed andtumors harvested for evaluation of protein expression by westernblotting analyses and immunohistochemistry. All animal procedures wereperformed according to the guidelines of the Canadian Council on AnimalCare and appropriate institutional certification.

Immunohistochemistry:

Immunohistochemical stains were performed on formalin-fixed andparaffin-embedded 4 μm sections of tumor samples using adequate primaryantibody, and the Ventana autostainer Discover XT (Ventana MedicalSystem) with enzyme labeled biotin streptavidin system and solventresistant 3,3′-diaminobenyidine Map kit. All comparisons of stainingintensities were made at 200× magnifications.

Statistical Analysis:

All in vitro data were assessed using the Student t test andMann-Whitney test. Tumor volumes of mice were compared usingKruskal-Wallis test. Overall survival was analyzed using Kaplan-Meiercurves and statistical significance between the groups was assessed withthe log-rank test (Graphpad Prism). Levels of statistical significancewere set at P<0.05.

Antibodies Used for Western Blotting:

PARP (1/1000) Caspase 3 (1/1000), Akt (1/1000), p-Akt (1/500), are fromcell signaling. Cyclin D1 (1/1000), HSP90 (1/1000), HSP70 (1/1000),clusterin (1/1000), AR (1/1000), PSA (1/1000) HSF-1 (1/1000) are fromSanta Cruz. HSP27 (1/5000) is from Assays Designs.

TABLE 3  Primers used for quantitative real-time PCT SequenceSequence 5′ to 3′ Sequence 5′ to 3′ name forward reverse ClusterinGAGCAGCTGAACGAGCAGT CTTCGCCTTGCGTGAGGT TT (SEQ ID NO: 45)(SEQ ID NO: 46) Hsp70 TGCCCTATCCAGATCCTGC GAGCCATCAGACTGAGGAGTA (SEQ ID NO: 47) TGA (SEQ ID NO: 48) Hsp90 TTCAGGCCCTTCCCGAATTCACTCCTTCCTTGGCAAC (SEQ ID NO: 49) AT (SEQ ID NO: 50)

DISCUSSION

Prostate cancer responds initially to anti-androgen therapies, however,progression of castration resistant disease frequently occurs. Smallmolecule inhibitors of Hsp90 show promise in the treatment ofcastration-resistant prostate cancer (CRPC) and other cancers, howeverthese inhibitors trigger a heat shock response that attenuates drugeffectiveness. In prostate cancer, treatment resistance emerges earlydue to compensatory mechanisms involving activation of heat shock factor1 (HSF-1). Once released from Hsp90, HSF-1 translocates to the nucleus,binds to heat shock elements (HSE) of Hsp genes and increases Hsptranscription activity (Whitesell et al., 2005). Therefore, Hsp90inhibition induces a heat shock response with increased expression ofseveral Hsps including Hsp90, Hsp70, Hsp27 and clusterin (CLU), whichenhance tumor cell survival and treatment resistance. The up-regulationof these molecular chaperones has been reported to play a role incellular recovery from stress by restoring protein homeostasis,promoting thermotolerance, cell survival, and treatment resistance(Takayama et al., 2003; Zoubeidi et al., 2010). The data herein showthat preventing CLU induction in this response would enhance Hsp90inhibitor-induced CRPC cell death in vitro and in vivo. As disclosedherein, CRPC was treated with Hsp90 inhibitor HSP90i-2-PRO or HSP90i-1in the absence or presence of custirsen, an antisense drug that targetsCLU. Treatment with either Hsp90 inhibitor alone increased nucleartranslocation and transcriptional activity of the heat shock factorHSF-1, which stimulated dose- and time-dependent increases in heat shockprotein expression, including especially CLU expression.Treatment-induced increases in CLU were blocked by custirsen, such thatthe combination of custirsen and either Hsp90 inhibitor had enhancedinhibition activity on CRPC cell growth and apoptosis compared tocustirsen or Hsp90 inhibitor monotherapy. Accompanying these effects wasa decrease in HSF-1 transcriptional activity as well as expression ofHSPs, Akt, PSA and androgen receptor. In vivo evaluation of the Hsp90inhibitors with custirsen in xenograft models of human CRPC demonstratedthat custirsen markedly potentiated anti-tumor efficacy, leading to an80% inhibition of tumor growth with prolonged survival compared to Hsp90inhibitor monotherapy. Together, the findings herein indicate that Hsp90inhibitor-induced activation of the heat shock response and CLU isattenuated by custirsen, with combination therapy having increasedpotency on delaying CRPC progression.

Development of treatment resistance is a common feature of mostmalignancies and the underlying basis for most cancer deaths. Treatmentresistance evolves, in part, from selective pressures of treatment thatcollectively increase the apoptotic rheostat of cancer cells. Survivalproteins up-regulated after treatment stress include anti-apoptoticmembers of the bcl-2 protein family, survivin, and molecular chaperoneslike CLU and other HSPs (Zellweger et al. 2003).

Molecular chaperones help cells cope with stress-induced proteinaggregation, and play prominent roles in cell signaling andtranscriptional regulatory networks. Chaperones act as genetic buffersstabilizing the phenotype of various cells and organisms at times ofenvironmental stress, and enhance Darwinian fitness of cells duringcancer progression and treatment resistance (Whitesell et al., 2005).Heat shock chaperones are key components of the heat shock response, ahighly conserved stress-activated protective mechanism also associatedwith oncogenic transformation and thermotolerance (Dai et al., 2007).Chaperones are particularly important in regulating misfolded proteinand endoplasmic reticular (ER) stress responses, an emerging area ofinterest in treatment stress and resistance. A growing enthusiasm fortherapeutic modulation of this proteostasis network highlights Hsp's andCLU as rational targets because of their multifunctional roles insignaling and transcriptional networks associated with cancerprogression and treatment resistance. Cancer cells express higher levelsof molecular chaperones and pirate the protective functions of HSF1 tosupport their transformation (Dai et al., 2007). Indeed, inhibitors ofHsp90, Hsp70, Hsp27 or CLU have all been reported to induce cancer celldeath and sensitize chemotherapy (Lamoureux et al., 2011; Guo et al.,2005).

Increased expression of clusterin (CLU) has been associated withchemoresistance, radioresistance, and hormone resistance (Zellweger etal., 2003; July et al., 2004). CLU is a stress-induced cytoprotectivechaperone that inhibits protein aggregation in a manner analogous tosmall HSPs, and its promoter contains a 14-bp element recognized by thetranscription factor HSF-1 (Humphreys et al., 1999). In human PCa, CLUlevels are low in Gleason grade 3 untreated hormone-naive tissues, butincrease with higher Gleason score (Steinberg et al., 1997) and withinweeks after androgen deprivation (July et al., 2002). CLU expressioncorrelates with loss of the tumor suppressor gene Nkx3.1 during theinitial stages of prostate tumorigenesis in Nkx3.1 knockout mice (Songet al., 2009). Experimental and clinical studies associate CLU withdevelopment of treatment resistance, where CLU suppressestreatment-induced cell death in response to androgen withdrawal,chemotherapy or radiation (Miyake et al., 2000a; July et al., 2002;Miyake et al., 2000b; Miyake et al., 2000c). Over-expression of CLU inhuman prostate LNCaP cells accelerates progression after hormone- orchemo-therapy (Miyake et al., 2000a; Miyake et al., 2000c), identifyingCLU as an anti-apoptotic gene up-regulated by treatment stress thatconfers therapeutic resistance. Custirsen is a second-generationphosphorothioate antisense oligonucleotide currently in late stageclinical development that potently inhibits CLU expression and enhancesthe efficacy of anticancer therapies in various human cancers includingPCa (Zoubeidi et al., 2010, Gleave et al., 2005). While targeting CLUenhances the cytotoxic effects of chemotherapy and delays tumor growthin various human cancers including PCa (Miyake et al., 2005), a role forCLU has not been characterized in the context of Hsp90 inhibitortreatment and resistance. As shown herein, Hsp90 inhibition induces aheat shock response with increased HSF-1 activity and CLU expression,which functions to inhibit treatment-induced apoptosis and enhanceemergence of treatment resistance. Knockdown of CLU using custirsenpotentiates the effect of Hsp90 inhibitors in CRPC.

Aspects of the present invention relate to the unexpected discovery thatan oligonucleotide targeting clusterin expression such as custirsen,together with a Hsp90 inhibitor is a potent combination for treatment ofprostate cancer. The discovery that an ant-clusterin therapy combinedwith Hsp90 is so potent is particularly surprising because Hsp90 isknown to increase the expression of multiple cytoprotective proteins.

Several Hsp90 inhibitors including HSP90i-2 have potent anti-tumoractivity in various preclinical models (Lamoureux et al., 2001;Chandarlapaty et al., 2008; Okawa et al., 2009) and are in clinicaltrials (Lamoureux et al., 2011; Sydor et al., 2006). Consistent withprior reports (Lamoureux et al., 2011; Cervantes-Gomez et al., 2009),the data herein show that Hsp90 inhibitors induce a stress response withactivation of the transcription factor HSF-1 and subsequent increasedlevels of Hsp90 itself, Hsp70 and CLU. This heat shock response likelyenhance emergence of treatment resistance, as inhibition oftranscription using Actinomycin D attenuates HSP90i-1-mediated Hsp70 andHsp27 expression and potentiates the effect of HSP90i-1 in vitro(Cervantes-Gomez et al., 2009). Additionally, inhibition of the stressresponse by silencing HSF-1 also increases the activity of Hsp90inhibitors (Bagatell et al., 2000). The experiments disclosed hereinevaluated the role of CLU in this heat shock response since CLU isdramatically induced by Hsp90 inhibitor treatment and CLU inhibitors arein late stage clinical development.

CLU is associated with many varied patho-physiological processesincluding reproduction, lipid transport, complement regulation andapoptosis (Zoubeidi et al. 2010; Rosenberg et al., 1995). CLU expressionis rapidly upregulated in various tissues undergoing apoptosis,including normal and malignant prostate and breast tissues followinghormone withdrawal (Kyprianou et al., 1990; Kyprianou et al., 1991).Previous studies have also linked CLU expression with induction andprogression of many cancers, including CRPC (Zoubeidi et al., 2010).Furthermore, CLU up-regulation following androgen ablation in xenografttumor models accelerates progression to castrate resistance and renderscells resistant to other apoptotic stimuli, including taxanechemotherapy (Miyake et al., 2000; Miyake et al., 2001). Consistent withthese accumulated findings (Miyake et al., 2001), inhibition of CLUusing custirsen synergistically enhances conventional as well asmolecular targeted therapies in PCa preclinical models (Sowery et al.,2008). Indeed, custirsen is now in Phase III trials as Phase II studiesreported >90% inhibition of CLU in human prostate cancer tissues (Chi etal., 2005), and 7 months prolonged survival when OGX-011 is combinedwith docetaxel in CRPC (Chi et al., 2008; Chi et al., 2010).

The data herein show that Hsp90 inhibitors increase CLU levels both invitro and in vivo, while clusterin inhibits HSP90i-2 or HSP90i-1 inducedCLU. As expected (Cervantes-Gomez et al., 2009; Bagatell et al., 2000),HSP90i-2 or HSP90i-1 induces HSF-1 transcriptional activity leading toup-regulation of HSPs expression. Surprisingly, the experimentsdescribed herein found that CLU silencing abrogates, while CLUoverexpression enhances, Hsp90 inhibitor-induced HSF-1 transcriptionactivity, identifying a role for CLU in the regulation of HSF-1 and theheat shock response itself. CLU knockdown blocks the translocation toHSF-1 to the nucleus following treatment with Hsp90 inhibitors. Thiseffect of CLU on HSF-1 activity is biologically relevant since CLUoverexpression protects, while CLU silencing enhances, cytotoxicity ofHsp90 inhibitors. Consistent with these in vitro results, synergisticeffects were also observed in vivo in PC-3 and LNCaP models whencustirsen was combined with Hsp90 inhibitors. Combination custirsen plusHsp90 inhibitor significantly delay CRPC tumor growth and prolongedsurvival in PC-3 and LNCaP models. Increased apoptotic rates withcombined Hsp90 and CLU inhibition suggests that delayed tumorprogression resulted from enhanced treatment-induced apoptosis. Systemicadministration of an oligonucleotide which reduces clusterin expressionplus a Hsp90 inhibitor decreases tumor growth compared with control ScrBASO plus an Hsp90 inhibitor in PC-3 model and LNCaP castration-resistantprostate cancer, respectively. This inhibition of tumor progression isaccompanied with a prolongation of survival in both prostate cancermodels. Detection of increased apoptosis after combined clusterin plusHsp90 inhibition by detection of TUNEL using immunohistochemistrysuggests that delayed tumor progression after combined therapy resultsfrom enhanced Hsp90 inhibitor-induced apoptosis. Collectively, theseresults highlight, for the first time, a biologically relevantfeed-forward regulation loop of CLU on HSF-1 and the heat shockresponse.

The effect of an oligonucleotide which reduces clusterin expression incombination with an Hsp90 inhibitor on PSA level was examined in theLNCaP castration-resistant prostate cancer model as disclosed hereinabove. As shown herein, targeting CLU using siRNA or the antisense drug,custirsen, suppressed treatment-induced CLU induction and enhanced Hsp90inhibitor-induced cell death in prostate cancer cells. Serum PSA levelis an established and useful biomarker regulated by androgen receptor(AR) in the presence of androgens (Magklara et al., 2002), and avaluable tool in the follow-up of patients to assess the efficacy ofchemotherapy. In addition to the effects of CLU inhibition on the heatshock response, observations in the castrate-sensitive, AR-positiveLNCaP model highlight another possible benefit of combined CLU and Hsp90suppression involving AR activity. Hsp90 inhibition is known todestabilize and degrade the AR with decreased PSA expression (Solit etal., 2002; Georget et al., 2002). In vivo, serum PSA levels as well asPSA doubling time and velocity, were significantly reduced withcombination OGX-011 therapy compared with PF-04929113 monotherapy. SerumPSA level is an established and useful AR-regulated biomarker (Kim etal., 2004) and a valuable tool in assessing efficacy of chemotherapy.Interestingly, at the low doses of Hsp90 inhibitor used in this in vivostudy, no effect on serum PSA level was apparent. Lower PSA levels withcombination therapy correlated with lower AR levels. This correlationbetween CLU inhibition and lower AR levels may involve the regulationloop of CLU on HSF-1 and the role of HSF-1 in regulating expression ofother AR chaperones (eg. Hsp27, Hsp70, Hsp90, FKBP5.2) and we areactively exploring the molecular basis in ongoing experiments. While CLUis known to be transcriptionally activated by HSF-1 (Zoubeidi et al.,2010), the data herein also show that CLU exerts a feed forward loopthat in turn activates HSF-1. CLU knockdown decreases HSF-1transcriptional activity and abrogates its nuclear translocation, whichsubsequently leads to decreased Hsp27, Hsp70 and Hsp90 expression,similar to that observed after HSF-1 knockdown (Rossi et al., 2006).Consequently, AR stability is reduced because of lowered chaperonelevels.

In addition to increased potency of anti-tumor activity, combinationtherapy may also allow dose reduction strategies to reduce toxicity. Forexample, HSP90i-1 induced hepatotoxicity as monotherapy at 60 mg/kg/day(Glaze et al., 2005), while HSP90i-2-PRO caused body weight loss at 50mg/kg/day. In a previous study, 50 mg/kg HSP90i-2-PRO as monotherapyinhibited LNCaP CRPC tumor progression (Lamoureux et al., 2011). Atsub-therapeutic doses of mg/kg/day used in the present study,HSP90i-2-PRO monotherapy showed marginal, non-significant decreases intumor volume and no effect on serum PSA levels; however, significantdelays in tumor progression were seen at this lower dose whenHSP90i-2-PRO was combined with custirsen, with no toxicity observed.

The data disclosed herein help define how stress induced by Hsp90inhibitors regulates CLU by induction of HSF-1 activity and, in turn,how CLU regulates HSF-1 activity, cell survival, and treatmentresistance. As demonstrated herein, for the first time, that CLUinhibition abrogates the heat shock response induced Hsp90 inhibitors.These observations are clinically relevant since CLU inhibitors are inphase III clinical trials, and provide a framework for building new drugcombinations based on mechanism-based interventions to overcome drugresistance. The present invention relates to the development of targetedstrategies employing custirsen in combination with Hsp90 inhibitors toimprove patient outcome in CRPC.

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1-33. (canceled)
 34. A pharmaceutical composition comprising an amountof an oligonucleotide which reduces clusterin expression, and a Hsp90inhibitor, wherein the oligonucleotide is an antisense or RNAioligonucleotide the reduces clusterin expression, and the Hsp90inhibitor is4-(6,6-Dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-2-(4-hydroxy-cyclohexylamino)-benzamide,or a pharmaceutically acceptable salt thereof, or a prodrug that ismetabolized to release4-(6,6-Dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-2-(4-hydroxy-cyclohexylamino)-benzamide.35. The pharmaceutical composition of claim 34, wherein theoligonucleotide is an antisense oligonucleotide.
 36. The pharmaceuticalcomposition of claim 35, wherein the antisense oligonucleotide comprisesone of SEQ ID NOs 3, 4 and
 11. 37. The pharmaceutical composition ofclaim 36, wherein the antisense oligonucleotide comprises SEQ ID NO: 3.38. The pharmaceutical composition of claim 37, wherein the antisenseoligonucleotide is modified to enhance in vivo stability relative to anunmodified oligonucleotide of the same sequence.
 39. The pharmaceuticalcomposition of claim 38, wherein the oligonucleotide is custirsen. 40.The pharmaceutical composition of claim 39, wherein the Hsp90 inhibitoris Hsp90i-2-PRO.
 41. The pharmaceutical composition of claim 37, whereinthe Hsp90 inhibitor is Hsp90i-2-PRO.
 42. The pharmaceutical compositionof claim 35, wherein the Hsp90 inhibitor is Hsp90i-2-PRO.
 43. Thepharmaceutical composition of claim 34, wherein the Hsp90 inhibitor isHsp90i-2-PRO.